Fig 2: TG2-dependent chemokine production in keratinocytes is critical for IL-17-producing CCR6+ ?dT-cell and neutrophil dermal infiltration.a, b Effect of DMSO- or IMQ-treated WT- or TG2-/--CM on CCR6+ ?dT-cell (a) and CD11b+ Ly-6G+ cell (b) migration according to Transwell migration assays. Flow cytometric analysis of the percentage of cells migrating toward the CM in the presence or absence of anti-CXCL1 (a) and anti-CCL20 (b) antibodies (n = 3/group). c, d WT and TG2-/- mouse-derived CCR6+ ?dT-cell (c) and CD11b+ Ly-6G+ cell (d) migration in response to recombinant mouse CCL20 and CXCL1, respectively. Data represent the mean ± SEM (n = 3/group). *p < 0.05; **p < 0.01. e, f Cells were isolated from the back skin of WT and TG2-/- mice treated with IMQ for four consecutive days and analyzed by flow cytometry. Representative flow cytometric profiles of CCR6+ ?dTCRlow cells (e) and CD11bhigh Ly-6G+ cells (f). The percentage of cells is shown. Data represent the mean ± SEM (n = 9/group). g Skin sections were immunostained with CCR6 (green)- and IL-17 (red)-specific antibodies. Nuclei were stained with DAPI (blue). Scale bar, 100 µm. h–j Back skin was separated into epidermis and dermis and analyzed by RT-PCR for IL-17-producing ?dT-cell markers (h) (Ccr6 and Il23r), IL17 and related cytokines (i) (Il17a, Il17f, and Il22), and myeloid cell-derived cytokines (j) (Il12a and Il23a). Data represent the mean ± SEM (n = 11/group). *p < 0.05; **p < 0.01 vs. WT mice.

Fig 3: TKO Tregs Display Marked Defects in Mobilization In Vitro and In Vivo(A) CD4+CD25- T cells from spleens of WT and TKO mice after 12 weeks of HFD were treated with anti-CD3 antibodies and TGF-ß1 for differentiation into in vitro differentiated Tregs (iTregs). Percentage of WT and TKO CD4+CD25+Foxp3+ Tregs were measured by flow cytometry at the indicated time points (n = 6 per group). (B and C) CD4+CD25- T cells from spleens of WT and TKO mice after 12 weeks of HFD were activated by anti-CD3 antibodies for 24 h and subjected to qRT-PCR analysis (B) or ELISA from supernatants (C) for the indicated cytokines, chemokines, and growth factors (n = 5–9 per group).(D and E) Transwell migration study of CD4+CD25+ Tregs isolated from WT and TKO mice after 12 weeks of HFD. Cells were assessed for migration in the presence or absence of CCL19 (D) or CCL20 (E) (n = 3 per group).(F and G) Flow cytometry for CCR7 (F) or CCR6 (G) expression in WT and TKO Tregs (n = 6 per group).(H) Schematic of PKH26-labeled HFD WT and TKO Tregs adoptively transferred to HFD C57BL/6 mice. Flow cytometry shows percentage of PKH26-expressed cells in liver, VAT, and SAT of recipient mice (n = 6 per group).(I and J) Schematic of glucose uptake study of differentiated 3T3-L1 cells co-cultured with HFD WT and TKO iTreg supernatant (supe) (I). (J) Fluorescence intensity of 2-Deoxy-D-glucose (2-DG) uptake by differentiated 3T3-L1 cells co-cultured with supernatants of WT and TKO CD4+ Tregs in the presence or absence of insulin stimulation (n = 4 per group).(K and L) Schematic of glucose production study of mouse primary hepatocytes co-cultured with HFD WT and TKO iTreg supernatants (K). (L) Glucose production by mouse primary hepatocytes co-cultured with supernatants of HFD WT and TKO CD4+ Tregs (n = 6 per group).Statistical differences are indicated as *p<0.05, **p<0.01, and ***p<0.001. Results are reported as mean ± SEM.Related to Figures S3 and S4.

Fig 4: Commensal Bacteria Are Required for Mincle-Dependent Th17 Generation in PPs(A and B) Frequency of IL-17 production by intracellular staining in re-stimulated CD4+ T cells from PPs of the indicated genotypes after treatment with an antibiotic cocktail (ABX) containing ampicillin, neomycin, metronidazole, and vancomycin (A) or vancomycin only (B) in the drinking water over 4 weeks.(C) Frequency of IL-17 and IL-22 production by intracellular staining of re-stimulated OT-II T cells co-cultured with sorted WT and Mincle-deficient (Clec4e-/-) GM-DCs loaded with OVA323–339 peptide, as in Figures 1D and 1E; the cells were pulsed or not (medium) with mucosal-associated commensals from WT mice treated with ABX during gestation and lactation and gavaged with L. plantarum at weaning as indicated (L. plantarum-enriched mucus; 10:1 DC ratio).(D) ELISA of IL-6 and IL-23 from the co-culture of GM-DC in (C).(E and F) WT littermates and Mincle-deficient (Clec4e-/-) mice were treated with ABX during gestation and lactation and were gavaged with L. plantarum (1 × 106) at weaning (+L. plantarum gavage) or not (WT), as indicated in the figure. The frequency of IL-17- and IL-22-producing re-stimulated CD4+ T cells (E) or CCR6+ ILCs (F) from PPs by intracellular staining in the indicated genotypes and conditions is shown.Data represent one representative experiment of two performed (A and F) or were pooled from at least two independent experiments (B–E). Each symbol represents an individual mouse. The arithmetic mean for each group is indicated. *p < 0.05, **p < 0.01, ***p < 0.001 (A–E: one-way ANOVA and Bonferroni post hoc test; F: unpaired two-tailed Student’s t test). See also Figure S5.

Fig 5: Females tend to modulate the hepatic inflammation better than males as they age on a regular chow diet(A) Female or male DIAMOND mice received a CD for 24 and 40 weeks, starting at 10 weeks of age (three to five mice/group). Livers were collected after 24 and 40 weeks of being on a CD and subjected to an H&E staining followed by enumeration of inflammatory cells using a machine learning (ML) model trained to detect and quantitate lobular inflammation on whole-slide histology images. ML lobular inflammation is shown as blue color.(B) FVS- viable cells gated for CD3+CD4+ T cells were analyzed for the percentage of Th1 (CXCR3+CCR5-), Th2 (CCR8+IL-3Ra-), or Th17 (CCR6+CD161-) cells: percentage of Th1 cells that do not express TIM-3 was determined.(C) FVS- viable cells were gated for CD3+ T cells to show the proportion of CD8+ T cells and CD4+ T cells in males or females being on a CD for 24 weeks (24MCD or 24FCD) and 40 weeks (40MCD or 40FCD), were analyzed for M1 (F4/80+CD68+CD206-) and M2 macrophages (F4/80+CD68+/-CD206+), and were analyzed for NK cells (CD3-CD4-CD8-CD49b+) and NKT cells (CD3+CD4-CD8-CD49b+). Percent total cells were analyzed for the ratio of CD8+ T cells to MDSC (CD8/MDSC) or CD4+ T cells to MDSC (CD4/MDSC).(D) FVS- viable splenic CD8+ or CD4+ T cells were analyzed for percentage of T cell phenotypes, including Te (CD44+CD62L-), Tem (blue line, CD44+CD62Llow), Tcm (red line, CD44+CD62Lhigh), and Tn (CD44-CD62L+) cells.Error bars are SEM. *p = 0.01, **p = 0.001, and ***p = 0.0001.